Protein transduction strategies are extremely useful for the investigation and manipulation of cellular processes. Proteins modified with fluorophores in vitro and delivered into live cells can, for instance, be used for imaging applications. In addition, the in cellulo determination of protein structure by nuclear magnetic resonance (NMR) has been achieved by delivering isotopically labeled proteins into live human cells. Furthermore, transcription factors that are rendered cell-permeable by tagging with cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) have emerged as potential tools for ex vivo tissue regeneration applications. For instance, the transcription factors Oct4, Sox2, and Klf4 labeled with 11R or 9R reprogram fibroblasts into induced pluripotent stem cells. The transcription factor HoxB4 tagged with the PTD TAT can also be used to expand hematopoietic stems cells in vitro and potentially increase the success rate of cell transplantation procedures. These protein delivery approaches are thought to represent a safer alternative than DNA-based strategies because proteins presumably do not alter the genomic integrity of cells and because their activity is lost upon proteolysis. Cells manipulated with proteins are, therefore, less likely to give rise to cancer after transplantation into patients.
While these studies illustrate the unique opportunities provided by protein transduction technologies, current protocols are often suboptimal. PTD-proteins typically utilize the endocytic pathway as a route of cellular entry. However, the majority of PTD-proteins endocytosed by cells typically remain trapped inside endosomes. As a result, the level of protein that reaches the cytosol of cells is low and the biological outcomes achieved are poor. A possible solution to this problem is to increase the ability with which proteins escape from the endocytic pathway. This is possible with membrane-destabilizing agents that disrupt endosomes. Protocols that combine endosomolytic agents and protein of interest have therefore been examined. CPPs modified with pH-activated sequences have, for instance, been reported as additives that can improve delivery of proteins in cis or in trans. However, the hydrophobicity of the membrane-active peptide is problematic and delivery efficiency remains low. Branched multimeric CPPs have also been used in a similar manner. A branched species containing three TAT copies causes the endosomal escape of different cargos with increased efficiency. In this case however, the complicated synthetic protocols required to generate such reagents are inconvenient. Furthermore, despite improving delivery, a concern associated with these membrane active reagents is cytotoxicity. The specificity of endosomolytic agents for endosomal membranes has not been clearly characterized and lysis of the plasma membrane of cells is often observed. Furthermore, the level of endosomal leakage that can be achieved without affecting cell viability has not been established.
Despite the advances in the art, a need remains for improved methods and reagents to facilitate delivery of a wide variety of molecules and reagents to the interior of living cells with high efficiency and low impact on the viability of the target cells. Specifically, a need remains for methods and reagents to facilitate endosomal escape of molecules and reagents applied to cells with a high efficiency, low toxicity, and convenience in protocols. The present disclosure addresses this need and provides further advantages related thereto.